bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.428777; this version posted February 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Spatial and temporal plasticity of neoantigen-specific T-cell responses bases on characteristics associated to antigen and TCR Authors Eva Bräunlein1*, Gaia Lupoli1*, Esam T. Abualrous2, Niklas de Andrade Krätzig3,4, Dario Gosmann1, Franziska Füchsl1, Lukas Wietbrock5, Sebastian Lange3,4,6, Thomas Engleitner3,4, Huan Lan2, Stefan Audehm1, Manuel Effenberger7, Melanie Boxberg8,9, Katja Steiger8,10,11, Yinshui Chang1, Kai Yu1, Cigdem Atay1,11, Florian Bassermann1,4,11, Wilko Weichert8,9,10,11, Dirk H. Busch7, Roland Rad3,4,6,11, Christian Freund2, Iris Antes5, Angela M. Krackhardt1,4,11‡ Affiliation 1 Technische Universität München, School of Medicine, Klinik und Poliklinik für Innere Medizin III, Klinikum rechts der Isar, Ismaningerstr. 22, Munich 81675, Germany. 2 Freie Universität Berlin, Laboratory of Protein Biochemistry, Institute for Chemistry & Biochemistry, Thielallee 63, 14195 Berlin, Germany 3 Technische Universität München, Institute of Molecular Oncology and Functional Genomics, TUM School of Medicine, Germany 4 Technische Universität München, Center for Translational Cancer Research (TranslaTUM), TUM School of Medicine, Germany 5 Technische Universität München, TUM School of Life Sciences and Center for Integrated Protein Science Munich, Emil-Erlenmeyer Forum 8, 85354 Freising, Germany. 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.428777; this version posted February 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 6 Technische Universität München, Department of Medicine II, Klinikum rechts der Isar, TUM School of Medicine, Munich, Germany 7 Technische Universität München, Institute for Medical Microbiology, Immunology and Hygiene, Trogerstr. 30, Munich 81675, Germany. 8 Technische Universität München, Institute of Pathology, School of Medicine, Ismaningerstr. 22, Munich 81675, Germany. 9 Technische Universität München, MRI-TUM-Biobank at the Institute of Pathology, School of Medicine, Ismaningerstr. 22, Munich 81675, Germany. 10 Technische Universität München, Core Facility Experimental Pathology, School of Medicine, Ismaningerstr. 22, Munich 81675, Germany. 11German Cancer Consortium (DKTK), partner-site Munich; and German Cancer Research Center (DKFZ) Heidelberg, Germany * Joined first co-authors Running Title Characterizing TCR candidates for adoptive T-cell transfer Keywords Cancer immunotherapy, adoptive T cell transfer, anti-tumor immune responses 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.428777; this version posted February 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Additional information: This work was supported by grants to A. M. Krackhardt from EIT Health (No 19638), Deutsche Krebshilfe (110281) and Wilhelm Sander-Stiftung (2015.030.1), as well as to A. M. Krackhardt and I. Antes from Deutsche Forschungsgemeinschaft (DFG) (SFB824/C10, SFB1035/A10 and CIPSM) and the Technical University of Munich and International Graduate School of Science and Engineering (IGSSE). Corresponding Author (‡) Angela M. Krackhardt, Klinik und Poliklinik für Innere Medizin III, Technische Universität München, School of Medicine, Ismaningerstr. 22, 81675 München, Tel. +49 89 4140 4124, Fax. +49 89 4140 4879, [email protected] Disclosure of Potential Conflicts of Interest The authors have declared that no conflict of interest exists. Word count: (From Introduction to Discussion) 5072 words 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.428777; this version posted February 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Statement of translational relevance Immunotherapy has demonstrated high efficacy in diverse malignancies. Neoantigens derived from mutations provide promising targets for safe and highly tumor-specific therapeutic approaches. Yet, single determinants of an effective and enduring T-cell mediated tumor rejection are still not well understood. We analyzed in detail seven neoantigen-specific T-cell receptors (TCRs) derived from a melanoma patient targeting three different altered peptide ligands identified by mass spectrometry and prediction analyses. Functional characterization of these TCRs revealed potent anti-tumor reactivity of all TCRs. Of special interest, TCRs with comparably lower affinity demonstrated effective in vivo activity as well as dominant spatial and temporal distribution in blood and tissue. Functional differences of TCR may require further T-cell and/or TCR engineering and should be considered for future clinical trial designs. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.428777; this version posted February 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Neoantigens derived from somatic mutations have been demonstrated to correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of personalized medicine approaches although their quality and associated immune responses is not yet well understood. In a case study of metastatic malignant melanoma, we performed an in-depth characterization of neoantigens and respective T-cell responses in the context of immunotherapy with Ipilimumab. Three neoantigens identified either by immunopeptidomics or in silico prediction were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient immune repertoire recognizing these antigens. TCRs were compared in-vitro and in-vivo with multi-parametric analyses. Identified immunogenic peptides showed similar binding affinities to the human leukocyte antigen (HLA) complex and comparable differences to their wildtype counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs differed substantially in functionality and frequency. In fact, TCRs with comparably lower functional avidity and higher potential for cross-reactivity provided at least equal anti-tumor immune responses in vivo. Of note, these TCRs showed a reduced activation pattern upon primary in vitro stimulation. Exploration of the TCR-β repertoire in blood and in different tumor-related tissues over three years, offered insights on the high frequency and particular long-term persistence of low-avidity TCRs. These data indicate that qualitative differences of neoantigen-specific TCRs and their impact on function and longevity need to be considered for neoantigen targeting by adoptive T-cell therapy using TCR-transgenic T cells. 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.428777; this version posted February 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction Cancer immunotherapy has demonstrated high efficacy in the treatment of diverse malignant diseases as shown by the efficacy of immune checkpoint modulation (1). These therapies can unleash anti-tumor immune responses, thereby conferring recognition and eradication of cancers by the patients’ own immune system without preexisting definition of targets or effector T-cell specificity. However, not all patients benefit from this therapeutic approach. Thus, understanding the nature of tumor recognition versus escape remains of fundamental importance to develop novel immunotherapeutic approaches and improve patients’ outcomes. The tumor mutational load is an important prognostic and predictive biomarker for therapeutic efficacy of immune checkpoint inhibitors across various disease entities, emphasizing the role of neoepitopes in tumor recognition and rejection (2). Therefore, identification and characterization of neoantigens are of particular interest for generation of new personalized immunotherapies, such as vaccination and cellular therapies (3-5). In order to fully exploit the potential of neoantigen-directed immunotherapy, more efforts are needed to understand qualities of neoepitopes and respective T-cell receptors (TCRs). We believe that case studies are currently a valid source to investigate the multitude of aspects involved (6) and that more detailed and comprehensive data are necessary to this purpose. We have previously identified two neoantigens by mass spectrometry (MS) and validated these by proof of defined neoantigen-specific autologous T-cell responses in a patient with melanoma (7). We have now extended the number of immunogenic antigens in this patient using additional prediction analyses and characterized neoantigens by 3D modeling as well as experimental validation of MHC-peptide stability. In addition, we identified seven TCRs recognizing these antigens. These TCRs were monitored in blood and different tissues of the patient over time and 6 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.02.428777; this version posted February 4, 2021.